Serine proteases have been shown to play a role in diverse physiological functions, many of which can play important roles in disease processes such as cardiovascular disease (Kohler, H. T., et al., (2000) N. Engl. J. Med. 342(24):1792-801; Hamsten, A., et al., (2000) Thromb. Haemost. 83(3):397403; Califf, R. M., et al., (2000) Circulation, 101(19):2231-8; Krendel, S., et al., (2000) Ann. Emerg. Med. 35(5):502-5), cancer (Schmidt, M., et al., (1999) Acta Otolaryngol. 119(8):949-53; Raigoso, P., et al., (2000) Int. J. Biol. Markers, 15(1):44-50; Haese, A., et al., (2000) J. Urol. 163(5):1491-7; Hoopera, J. D., et al., (2000) Biochim. Biophys. Acta, 2000, 1492(1):63-71; Wallrapp, C., et al., (2000) Cancer Res. 2000, 60(10):2602-6; Cao, Y., et al., (2000) Int. J. Mol. Med. 2000, 5(5):547-51), asthma, chronic obstructive pulmonary disease (COPD), inflammatory diseases (Rice, K. D., et al., (1998) Curr. Pharm. Des., (5):381-96; Nadel, J. A., et al., (1998) Eur. Respir. J., (6):1250-1; Wright, C. D., et al., (1999) Biochem. Pharmacol., 58(12):1989-96; Burgess, L. E., et al., (1999) Proc. Natl. Acad. Sci. U.S.A., 96(15):8348-52; Barnes, P. J., et al., (2000) Chest, 117(2 Suppl):1OS-4S) and bacterial infections (Al-Hasani, K., et al., (2000) Infect. Immun., 68(5):2457-63; Gaillot, O., et al., (2000) Mol Microbiol. 35(6):1286-94; Lejal, N., et al., (2000) J. Gen. Virol., 81(Pt 4):983-92).
Accordingly, serine protease activities of leukocyte elastase, thrombin, tissue plasminogen activator (t-PA) and plasmin are considered as potential useful markers for the diagnosis of some of these pathologies. For example, the tissue pathology of serine proteases is linked to their ability to be retained within diseased tissue, therefore providing molecular targets for in situ molecular imaging. However there remains a need in the art for new approaches of the molecular imaging of serine proteases in tissues. Serine protease molecular imaging agents that are easy and relatively cheap to produce are therefore particularly desirable.
Leukocyte elastase, thrombin, tissue plasminogen activator (t-PA) and plasmin belong to the S1 family of serine proteases, whose the mechanism of action is well known (Barrett, Handbook of Proteolytic Enzymes, 2nd Ed). The catalytic activity of the S1 family of proteases is provided by a charge relay system involving an aspartic acid residue that is hydrogen-bonded to a histidine, which itself is hydrogen-bonded to a serine. The sequences in the vicinity of the active site serine and histidine residues are well conserved in this family of proteases. The specificity of these proteases is essentially ensured by the “nonprime” domain of their active site i.e. the S5 or S4 to S1 binding subsites (following the nomenclature of Schechter and Berger (Schechter I., and Berger A. (1967) Biochem Biophys Res Commun 27, 157-162)) In these serine proteases, the amino acid interacting with the S1 subsite is either an arginine residue (t-Pa, Thrombin, Plasmin) or an hydrophobic residue (Leukocyte elastase)
Various irreversible ligands of the selected serine proteases are reported in the literature (Teger-Nilsson, 1977), (Kettner, 1978) (see table 1), They are constituted by small peptides interacting with the S5 or S4-S2 domain of the peptidase, and the amino acid binding the S1 subsite is replaced by an aminoacyl chloromethylketone (cmk) which interacts covalently with the serine of the catalytic site. Consequently, due to the restricted selectivity of the P1 residue, some of these irreversible inhibitors do not possess the required parameters of affinity and selectivity needed for a specific labelling of each protease. For example: in table 1, it was observed that the same peptide is described for Thrombin and t-PA.
TABLE 1Examples of irreversible ligands of the selected serine proteasesSerineproteasePeptideReferenceLeukocyteMeOSucAla-Ala-Pro-Ala-cmkNavia, PNAS, (1989), 86elastaseMeOSucAla-Ala-Pro-Val-cmkPowers, Biochim BiophysActa (1977), 485, 156tPAD-Phe-Pro-Arg-cmkBoatman, J Med Chem(1999), 42, 1367PlasminD-Val-Phe-Lys-cmkWoessner, Steroids(1989), 54, 491ThrombinD-Phe-Pro-Arg-cmkKettner, Thromb Res(1979), 14, 969
However, as shown in table 1, the ligand D-Val-Phe-Lys-COCH2Cl (dVFK-cmk, M.W. 500) was developed as a very selective irreversible peptide ligand of plasmin active site with high affinity (Collen, Biochimica and Biophysica Acta, 165 (1980), 158-166).
Moreover, no investigations have been carried out to use these irreversible ligands for the molecular imaging of serine proteases.